Modifications of the endosomal compartment in fibroblasts from sporadic Alzheimer's disease patients are associated with cognitive impairment.

Morphological alterations of the endosomal compartment have been widely described in post-mortem brains from Alzheimer's disease (AD) patients and subjects with Down syndrome (DS) who are at high risk for AD. Immunostaining with antibodies against endosomal markers such as Early Endosome Antigen 1 (EEA1) revealed increased size of EEA1-positive puncta. In DS, peripheral cells such as peripheral blood mononuclear cells (PBMCs) and fibroblasts, share similar phenotype even in the absence of AD. We previously found that PBMCs from AD patients have larger EEA1-positive puncta, correlating with brain amyloid load. Here we analysed the endosomal compartment of fibroblasts from a very well characterised cohort of AD patients (IMABio3) who underwent thorough clinical, imaging and biomarkers assessments. Twenty-one subjects were included (7 AD with mild cognitive impairment (AD-MCI), 7 AD with dementia (AD-D) and 7 controls) who had amyloid-PET at baseline (PiB) and neuropsychological tests at baseline and close to skin biopsy. Fibroblasts isolated from skin biopsies were immunostained with anti-EEA1 antibody and imaged using a spinning disk microscope. Endosomal compartment ultrastructure was also analysed by electron microscopy. All fibroblast lines were genotyped and their AD risk factors identified. Our results show a trend to an increased EEA1-positive puncta volume in fibroblasts from AD-D as compared to controls (p.adj = 0.12) and reveal enhanced endosome area in fibroblasts from AD-MCI and AD-AD versus controls. Larger puncta size correlated with PiB retention in different brain areas and with worse cognitive scores at the time of biopsy as well as faster decline from baseline to the time of biopsy. Finally, we identified three genetic risk factors for AD (ABCA1, COX7C and MYO15A) that were associated with larger EEA1 puncta volume. In conclusion, the endosomal compartment in fibroblasts could be used as cellular peripheral biomarker for both amyloid deposition and cognitive decline in AD patients.

Agomelatine: a new opportunity to reduce neuropathic pain-preclinical evidence.

Antidepressants are first-line treatments of neuropathic pain but not all these drugs are really effective. Agomelatine is an antidepressant with a novel mode of action, acting as an MT1/MT2 melatonergic receptor agonist and a 5-HT2C receptor antagonist that involves indirect norepinephrine release. Melatonin, serotonin, and norepinephrine have been involved in the pathophysiology of neuropathic pain. Yet, no study has been conducted to determine agomelatine effects on neuropathic pain in animal models. Using 3 rat models of neuropathic pain of toxic (oxaliplatin/OXA), metabolic (streptozocin/STZ), and traumatic (sciatic nerve ligation/CCI [chronic constriction nerve injury]) etiologies, we investigated the antihypersensitivity effect of acute and repeated agomelatine administration. We then determined the influence of melatonergic, 5-HT2C, α-2 and ß-1/2 adrenergic receptor antagonists in the antihypersensitivity effect of agomelatine. The effect of the combination of agomelatine + gabapentin was evaluated using an isobolographic approach. In STZ and CCI models, single doses of agomelatine significantly and dose dependently reduced mechanical hypersensitivity. After daily administrations for 2 weeks, this effect was confirmed in the CCI model and agomelatine also displayed a marked antihypersensitivity effect in the OXA model. The antihypersensitivity effect of agomelatine involved melatonergic, 5-HT2C, and α-2 adrenergic receptors but not beta adrenoceptors. The isobolographic analysis demonstrated that the combination of agomelatine + gabapentin had additive effects. Agomelatine exerts a clear-cut antihypersensitivity effect in 3 different neuropathic pain models. Its effect is mediated by melatonergic and 5-HT2C receptors and, although agomelatine has no affinity, also by α-2 adrenergic receptors. Finally, agomelatine combined with gabapentin produces an additive antihypersensitivity effect.

Chronic agomelatine treatment corrects the abnormalities in the circadian rhythm of motor activity and sleep/wake cycle induced by prenatal restraint stress in adult rats.

Agomelatine is a novel antidepressant acting as an MT1/MT2 melatonin receptor agonist/5-HT2C serotonin receptor antagonist. Because of its peculiar pharmacological profile, this drug caters the potential to correct the abnormalities of circadian rhythms associated with mood disorders, including abnormalities of the sleep/wake cycle. Here, we examined the effect of chronic agomelatine treatment on sleep architecture and circadian rhythms of motor activity using the rat model of prenatal restraint stress (PRS) as a putative 'aetiological' model of depression. PRS was delivered to the mothers during the last 10 d of pregnancy. The adult progeny ('PRS rats') showed a reduced duration of slow wave sleep, an increased duration of rapid eye movement (REM) sleep, an increased number of REM sleep events and an increase in motor activity before the beginning of the dark phase of the light/dark cycle. In addition, adult PRS rats showed an increased expression of the transcript of the primary response gene, c-Fos, in the hippocampus just prior to the beginning of the dark phase. All these changes were reversed by a chronic oral treatment with agomelatine (2000 ppm in the diet). The effect of agomelatine on sleep was largely attenuated by treatment with the MT1/MT2 melatonin receptor antagonist, S22153, which caused PRS-like sleep disturbances on its own. These data provide the first evidence that agomelatine corrects sleep architecture and restores circadian homeostasis in a preclinical model of depression and supports the value of agomelatine as a novel antidepressant that resynchronizes circadian rhythms under pathological conditions.

The novel antidepressant agomelatine normalizes hippocampal neuronal activity and promotes neurogenesis in chronically stressed rats.

Agomelatine is a novel antidepressant which acts as a melatonergic (MT1/MT2) receptor agonist and serotonergic (5-HT2C) receptor antagonist. The antidepressant properties of agomelatine have been demonstrated in animal models as well as in clinical studies. Several preclinical studies reported agomelatine-induced effects on brain plasticity, mainly under basal conditions in healthy animals. Yet, it is important to unravel agomelatine-mediated changes in the brain affected by psychopathology or exposed to conditions that might predispose to mood disorders. Since stress is implicated in the etiology of depression, it is valid to investigate antidepressant-induced effects in animals subjected to chronic stress. In this context, we sought to determine changes in the brain after agomelatine treatment in chronically stressed rats. Adult male rats were subjected to footshock stress and agomelatine treatment for 21 consecutive days. Rats exposed to footshock showed a robust increase in adrenocorticotropic hormone (ACTH) and corticosterone. Chronic agomelatine treatment did not markedly influence this HPA-axis response. Whereas chronic exposure to daily footshock stress reduced c-Fos expression in the hippocampal dentate gyrus, agomelatine treatment reversed this effect and normalized neuronal activity to basal levels. Moreover, chronic agomelatine administration was associated with enhanced hippocampal cell proliferation and survival in stressed but not in control rats. Furthermore, agomelatine reversed the stress-induced decrease in doublecortin expression in the dentate gyrus. Taken together, these data show a beneficial action of agomelatine in the stress-compromised brain, where it restores stress-affected hippocampal neuronal activity and promotes adult hippocampal neurogenesis.

Mechanisms contributing to the phase-dependent regulation of neurogenesis by the novel antidepressant, agomelatine, in the adult rat hippocampus.

Agomelatine is a novel antidepressant acting as a melatonergic receptor agonist and serotonergic (5-HT(2C)) receptor antagonist. In adult rats, chronic agomelatine treatment enhanced cell proliferation and neurogenesis in the ventral hippocampus (VH), a region pertinent to mood disorders. This study compared the effects of agomelatine on cell proliferation, maturation, and survival and investigated the cellular mechanisms underlying these effects. Agomelatine increased the ratio of mature vs immature neurons and enhanced neurite outgrowth of granular cells, suggesting an acceleration of maturation. The influence of agomelatine on maturation and survival was accompanied by a selective increase in the levels of BDNF (brain-derived neurotrophic factor) vs those of VEGF (vascular endothelial factor) and IGF-1 (insulin-like growth factor 1), which were not affected. Agomelatine also activated several cellular signals (extracellular signal-regulated kinase1/2, protein kinase B, and glycogen synthase kinase 3beta) known to be modulated by antidepressants and implicated in the control of proliferation/survival. Furthermore, as agomelatine possesses both melatonergic agonist and serotonergic (5-HT(2C)) antagonist properties, we determined whether melatonin and 5-HT(2C) receptor antagonists similarly influence cell proliferation and survival. Only the 5-HT(2C) receptor antagonists, SB243,213 or S32006, but not melatonin, mimicked the effects of agomelatine on cell proliferation in VH. The promoting effect of agomelatine on survival was not reproduced by the 5-HT(2C) receptor antagonists or melatonin alone. However, it was blocked by a melatonin antagonist, S22153. These results show that agomelatine treatment facilitates all stages of neurogenesis and suggest that a joint effect of melatonin agonism and 5HT(2C) antagonism may be involved in promotion by agomelatine of survival in the hippocampus.

The antidepressant agomelatine blocks the adverse effects of stress on memory and enables spatial learning to rapidly increase neural cell adhesion molecule (NCAM) expression in the hippocampus of rats.

Agomelatine, a novel antidepressant with established clinical efficacy, acts as a melatonin receptor agonist and 5-HT(2C) receptor antagonist. As stress is a significant risk factor in the development of depression, we sought to determine if chronic agomelatine treatment would block the stress-induced impairment of memory in rats trained in the radial-arm water maze (RAWM), a hippocampus-dependent spatial memory task. Moreover, since neural cell adhesion molecule (NCAM) is known to be critically involved in memory consolidation and synaptic plasticity, we evaluated the effects of agomelatine on NCAM, and polysialylated NCAM (PSA-NCAM) expression in rats given spatial memory training with or without predator stress. Adult male rats were pre-treated with agomelatine (10 mg/kg i.p., daily for 22 d), followed by a single day of RAWM training and memory testing. Rats were given 12 training trials and then they were placed either in their home cages (no stress) or near a cat (predator stress). Thirty minutes later the rats were given a memory test trial followed immediately by brain extraction. We found that: (1) agomelatine blocked the predator stress-induced impairment of spatial memory; (2) agomelatine-treated stressed, as well as non-stressed, rats exhibited a rapid training-induced increase in the expression of synaptic NCAM in the ventral hippocampus; and (3) agomelatine treatment blocked the water-maze training-induced decrease in PSA-NCAM levels in both stressed and non-stressed animals. This work provides novel observations which indicate that agomelatine blocks the adverse effects of stress on hippocampus-dependent memory and activates molecular mechanisms of memory storage in response to a learning experience.

NMDA receptor activation inhibits alpha-secretase and promotes neuronal amyloid-beta production.

Acute brain injuries have been identified as a risk factor for developing Alzheimer's disease (AD). Because glutamate plays a pivotal role in these pathologies, we studied the influence of glutamate receptor activation on amyloid-beta (Abeta) production in primary cultures of cortical neurons. We found that sublethal NMDA receptor activation increased the production and secretion of Abeta. This effect was preceded by an increased expression of neuronal Kunitz protease inhibitory domain (KPI) containing amyloid-beta precursor protein (KPI-APP) followed by a shift from alpha-secretase to beta-secretase-mediated APP processing. This shift is a result of the inhibition of the alpha-secretase candidate tumor necrosis factor-alpha converting enzyme (TACE) when associated with neuronal KPI-APPs. This KPI-APP/TACE interaction was also present in AD brains. Thus, our findings reveal a cellular mechanism linking NMDA receptor activation to neuronal Abeta secretion. These results suggest that even mild deregulation of the glutamatergic neurotransmission may increase Abeta production and represent a causal risk factor for developing AD.

Akt-dependent expression of NAIP-1 protects neurons against amyloid-{beta} toxicity.

Neurotrophins are a family of growth factors that attenuate several forms of pathological neuronal cell death and may represent a putative therapeutic approach to neurodegenerative diseases. In Alzheimer disease, amyloid-beta (Abeta) is thought to play a central role in the neuronal death occurring in brains of patients. In the present study, we evaluate the ability of neurotrophin-3 (NT-3) to protect neurons against the toxicity induced by aggregated Abeta. We showed that in primary cultures of cortical neurons, NT-3 reduces Abeta-induced apoptosis by limiting caspase-8, caspase-9, and caspase-3 cleavage. This neuroprotective effect of NT-3 was concomitant to an increased level of Akt phosphorylation and was abolished by an inhibitor of the phosphatidylinositol-3 kinase (PI-3K), LY294002. In parallel, NT-3 treatment reduced Abeta induced caspase-3 processing to control levels. In an attempt to link PI-3K/Akt to caspase inhibition, we evaluated the influence of the PI-3K/Akt axis on the expression of a member of the inhibitors of apoptosis proteins (IAPs), the neuronal apoptosis inhibitory protein-1. We demonstrated that NT-3 induces an up-regulation of neuronal apoptosis inhibitory protein-1 expression in neurons that promotes the inhibition of Abeta-induced neuronal apoptosis. Together, these findings demonstrate that NT-3 signaling counters Abeta-dependent neuronal cell death and may represent an innovative therapeutic intervention to limit neuronal death in Alzheimer disease.

Neurotrophin-3-induced PI-3 kinase/Akt signaling rescues cortical neurons from apoptosis.

A number of cytokines including neurotrophins have been tested for their neuroprotective activity against different paradigms of neuronal death. However, as for neurotrophin-3 (NT-3), their mechanisms of action have not been fully identified. By using cultures of mouse cortical neurons, we have investigated the molecular mechanisms by which neurotrophin-3 could protect cortical neurons against apoptosis. In a model of caspase-dependent apoptosis leading to the recruitment of active initiators caspase-8 and -9 and of executioner caspase-3, we have evidenced that NT-3 displayed an anti-apoptotic effect in a dose-dependent manner. First, we showed that, in cultured cortical neurons, NT-3 could promote extracellular signal-regulated protein kinase/mitogen-activated protein kinase (ERK/MAPK) and phosphatidylinositol-3' (PI-3) kinase/Akt phosphorylation. Second, we showed that although the blockade of the Akt pathway prevented the anti-apoptotic effect of NT-3, blockade of the ERK pathway did not. Altogether, our data demonstrate that NT-3 displayed an anti-apoptotic effect on cultured cortical neurons through a mechanism involving the recruitment of the PI-3 kinase/Akt signaling pathway.

Level of haem oxygenase does not obligatorily reflect the sensitivity of PC12 cells to an oxidative shock induced by glutathione depletion.

In order to investigate the function of haem oxygenase in neuronal cell death or survival, we have determined in PC12 cells whether induction of haem oxygenase mRNA and protein or inhibition of haem oxygenase activity may be able to modulate the cell response to an oxidative stress. Inhibition of glutathione biosynthesis by buthionine sulfoximine (BSO) has indeed been demonstrated, in this cell line, to decrease the intracellular content of glutathione and to trigger a gradual and programmed cell death. Inhibition of haem oxygenase by zinc protoporphyrin IX, a potent inhibitor of this enzyme, or by a recently described peptidic inhibitor, induced a significant decrease in the toxicity of BSO. This protective action was not due to an alteration in the metabolism of glutathione and was still observed when the protecting agent was added several hours after BSO treatment. Induction of haem oxygenase-1 mRNA and protein by either haemin or pyrrolidine dithiocarbamate was associated with no protection or a significant reduction in the toxicity of BSO respectively. Our results indicate that induction of haem oxygenase-1 is not obligatorily associated with an improved resistance towards oxidative stress and suggest that a byproduct of haem degradation may also become detrimental.